An induction medium and its application in inducing white peony root leaf begonia unpollinated ovule callus

Abstract

The present application relates to the field of callus induction, and particularly relates to a kind of induction medium and its application in the induction of white peony root leaf begonia unpollinated ovule callus, the induction medium includes basic medium, plant growth regulator, carbon source and coagulant;The basic medium is any one of B5 medium, Miller medium;The plant growth regulator includes TDZ, NAA and 6-BA;The concentration ratio of TDZ, NAA and 6-BA is TDZ:NAA:6-BA=(0.05-0.10):(0.25-1.00):(0.25-1.00);The pH value of the induction medium is 5.8-6.0.The present application establishes efficient white peony root leaf begonia callus induction system by adjusting the components of culture medium, and obtains embryoid and sprout by inducing callus redifferentiation, which lays a foundation for carrying out begonia haploid breeding research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of callus induction, in particular to a kind of induction medium and its application in inducing Begonia heracleifolia unpollinated ovule callus. BACKGROUND

[0002] Begonia heracleifolia is a plant of Begonia genus in Begoniaceae. The leaf shape and leaf spot of Begonia genus plant are very rich. The leaf of Begonia heracleifolia is palmate leaf, deeply lobed, and the leaf blade is different shades of green. The flower is pink oval, the inflorescence is erect and higher than the leaf surface, the flower quantity is large, the flowering period is long, and it has high ornamental value. It is also an important parent of modern American leaf Begonia.

[0003] Begonia genus plant is a very popular ornamental flower and leaf plant at home and abroad, and it is the fourth largest pot flower in the world. 10% of the varieties are applied in landscaping, and the horticultural output value is huge. It is regarded as one of the most valuable groups in the 21st century. In recent years, although new varieties of Begonia are increasing, it is still an important task to develop and promote Begonia flower industry to breed disease-resistant, adaptable and high ornamental value fine varieties.

[0004] At present, domestic Begonia breeding mostly uses conventional breeding methods, which has the disadvantages of long cycle and slow effect. However, haploid culture is an effective method to quickly obtain pure line plants, which provides the possibility to shorten the breeding period and improve the breeding efficiency. In haploid plants, recessive traits can be expressed, which enriches the breeding resources. In addition, the obtained haploid can be applied to gene transformation, mutagenesis, mutant screening and other researches, which provides a new way for germplasm innovation. In vitro androgenesis and gynogenesis of plants can obtain haploid, and unpollinated ovule culture is one of the important means to obtain haploid plants by in vitro gynogenesis. Unpollinated ovule culture is a special in vitro culture of sexual organs, which has the problems of high operation difficulty, low callus induction rate, and difficult callus differentiation compared with general tissue culture technology. At present, there is no related research report on in vitro gynogenesis of Begonia. Therefore, the establishment of unpollinated ovule callus induction and redifferentiation culture system of Begonia not only accumulates the foundation for obtaining haploid and double haploid materials of Begonia, but also is beneficial to the cultivation of new varieties of Begonia. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies in the prior art and provide a method for inducing unpollinated ovule callus of Begonia heracleifolia. By adjusting the components of the culture medium, a high-efficiency callus induction system of Begonia heracleifolia is established, and embryoids and seedlings are obtained by inducing callus redifferentiation, which lays a foundation for the research of haploid breeding of Begonia.

[0006] To achieve the above object, the technical scheme adopted by the present application is:

[0007] In the first aspect, the present application provides an induction medium, which comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; the basic medium is any one of B5 medium or Miller medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=(0.05-0.10):(0.25-1.00):(0.25-1.00); and the pH value of the induction medium is 5.8-6.0.

[0008] The culture of unpollinated ovules is a special in vitro culture of sexual organs, which has the problems of great operation difficulty, low callus induction rate, and difficult callus differentiation. By reasonably matching the components of the induction medium, the carbon source and the plant regulators, i.e. thidiazuron (TDZ), naphthalene acetic acid (NAA) and benzylaminopurine (6-BA), are added to the basic medium to meet the growth requirements of the callus. The plant growth regulators contained in the induction medium adopted by the present application can induce the callus of unpollinated ovules of Begonia fimbristipulata within the above-mentioned ratio range.

[0009] Preferably, the carbon source is sucrose; the coagulant is agar; and the weight ratio of the sucrose and the agar is sucrose:agar=30:8.

[0010] Preferably, the preparation method of the induction medium is as follows: the plant growth regulator, sucrose and agar are weighed according to the proportion, and the rest is supplemented with the basic medium; after the induction medium is prepared, high-pressure sterilization is performed under the conditions of a pressure of 105 Kpa and a temperature of 121℃ for 20 min, and after sterilization, the induction medium is divided into culture dishes on an ultra-clean bench.

[0011] Preferably, the basic medium is Miller medium.

[0012] Miller medium is a medium with medium inorganic salt content. Compared with other media, the amount of inorganic elements is reduced by 1 / 3-1 / 2, the types of trace elements are reduced, and inositol is not used. The callus induction rate obtained by using Miller medium for culture can reach 100%, which is significantly higher than that of MS medium and B5 medium, and the callus obtained by culture has a compact texture, which is more suitable for the culture of ovules of Begonia fimbristipulata.

[0013] Preferably, the concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.10:(0.5-1.00):(0.25-0.50).

[0014] The plant growth regulator contained in the induction medium is in the above-mentioned ratio range, and the induction rate of the un-pollinated ovule callus of Begonia fimbristipula is above 50%.

[0015] More preferably, the concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA = 0.10:1.00:0.50.

[0016] In the above-mentioned specific ratio, the un-pollinated ovule callus induction rate of Begonia fimbristipula is the highest after 6 days of low-temperature treatment, and the callus induction rate is 58.33%.

[0017] In the second aspect, the application provides the use of the above-mentioned induction medium in the induction of the un-pollinated ovule callus of Begonia fimbristipula.

[0018] In the third aspect, the application provides a method for inducing the un-pollinated ovule callus of Begonia fimbristipula, which comprises the following steps: inoculating the explant washed and sterilized into the above-mentioned induction medium, sealing the medium, and culturing to obtain the callus; the explant is a fresh Begonia fimbristipula female flower 1-3 days before opening.

[0019] Preferably, the Begonia fimbristipula female flower is picked up during 8 am to 14 pm; the selection condition is that the female flower is normal in appearance quality, free of diseases and pests, and 1-3 days before opening. The female flower grown at this stage has embryo sac cells close to maturity or maturity, and the induction effect is the best.

[0020] Preferably, the explant is pretreated before culturing; the pretreatment method is to place the explant at 4℃ for 2-6 days before washing or to place the sealed medium at 35℃ for 2-6 days for heat shock.

[0021] In order to change the physiological state, division mode and development path of the explant cells, the explant is usually pretreated to improve the callus induction rate and haploid plant regeneration rate; however, the effect of pretreatment varies according to different materials, which may show positive effect, negative effect or no influence. The induction method of the un-pollinated ovule callus of Begonia fimbristipula in the application is pretreated, which improves the differentiation rate of the un-pollinated ovule callus of Begonia fimbristipula and facilitates the subsequent plant regeneration culture.

[0022] Preferably, the explant is washed with sterile water; the sterilization process is to soak in ethanol first, then wash clean with sterile water, then soak in 2% sodium hypochlorite solution, and finally wash clean with sterile water.

[0023] The specific process of cleaning and disinfection is as follows: the female flower is placed in a sterile empty bottle, first washed with sterile water for 1-2 times, then soaked with 75% ethanol for 20s on the clean bench, washed with sterile water for 1-2 times, soaked with 2% sodium hypochlorite solution for 20min, and finally washed with sterile water for 5-6 times.

[0024] Preferably, the inoculation site of the explant is the ovule.

[0025] The separation and inoculation process of the ovule is as follows: the disinfected white peony leaf Begonia female flower is dried with sterile filter paper, the perianth of the female flower is cut off with a surgical knife, and then the ovary carpel is dissected off with a sharp forceps, and a lot of white powder-like substances on the embryo seat can be seen, which are the ovules. The ovules are clamped in the induction medium with a blunt forceps and rolled to make the ovules stick to the medium, and finally sealed with sealing film.

[0026] Preferably, the culture conditions are as follows: temperature 23-27℃, light time 16h / day, air relative humidity 30%-65%, and culture time 60-90 days.

[0027] The ovules can be obviously swollen and browned after 30-40 days of induction, the browned ovules are transferred to new induction medium in early stage, and basically no brown occurs after further culture, and the ovules restore normal growth. A small part of the ovules differentiate into embryos and sprouts after 60-90 days of culture.

[0028] The beneficial effects of the present application are as follows:

[0029] (1) In the selection of the basic medium, the induction rate of the Miller medium is obviously higher than that of the B5 medium and the MS medium. The ovules in the MS medium basically die with the increase of the culture time; the B5 medium can also induce callus, but the number of ovules in the callus induced by the culture dish is less; and the number of ovules in the callus induced by the culture dish in the Miller medium is large, the texture is tight, and the highest induction rate can reach 100%;

[0030] (2) The ovules of the Begonia plant are small (such as powder size), but the ovules can be easily separated without the aid of a dissecting microscope by using the induction method of the present application, and the step of pre-culturing the ovary slice and stripping the swollen ovule is also reduced;

[0031] (3) The ovules can be obviously swollen and browned after 30-40 days of induction, the browned ovules are transferred to new induction medium in early stage, and basically no brown occurs after further culture, and the ovules restore normal growth; after 60 days of culture on the callus induction and differentiation medium, a small part of the ovules differentiate into embryos and sprouts, and the callus subjected to heat shock for 6d has a higher differentiation rate;

[0032] (4) The application provides a method for efficiently inducing white peony leaf begonia unpollinated ovule callus, explores a key culture medium formula affecting white peony leaf begonia unpollinated ovule callus induction, establishes a high-efficiency white peony leaf begonia callus induction system, and successfully induces the obtained callus to differentiate into embryoids and seedlings, thereby laying a foundation for researches such as begonia plant ovule culture, haploid breeding, and mutagenic breeding. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is a schematic diagram of callus induction on different basic culture media; A is a schematic diagram after 60 days of inoculation on MS medium, B is a schematic diagram after 60 days of inoculation on B5 medium, and C is a schematic diagram after 60 days of inoculation on Miller medium.

[0034] Figure 2 It is an embryoid and a seedling differentiated from callus; A is an embryoid, and B is a seedling. DETAILED DESCRIPTION

[0035] In order to better illustrate the purpose, technical scheme and advantages of the application, the application will be further described below in combination with specific examples.

[0036] The formula of the MS medium, the B5 medium and the Miller medium used in the following experiments is shown in Tables 1-3 as follows:

[0037] Table 1 Composition of MS medium

[0038] Ingredients Content (mg / L) ammonium nitrate NH4NO3 1650 potassium nitrate KNO3 1900 Potassium phosphate monobasic KH2PO4 170 Magnesium sulfate heptahydrate MgS04-7H20 370 Calcium chloride dihydrate CaCl2-2H2O 440 Potassium iodide KI 0.83 boric acid H3BO3 6.2 Manganese sulfate tetrahydrate MnS04-4H20 22.3 Zinc sulfate heptahydrate ZnS04-7H20 8.6 Sodium molybdate dihydrate Na2Mo04-2H20 0.25 Copper sulfate pentahydrate CuSO4-5H2O 0.025 Cobalt chloride hexahydrate CoCl2.6H2O 0.025 Disodium ethylenediaminetetraacetate Na2·EDTA·2H2O 37.3 Ferrous sulfate heptahydrate FeSO4.7H2O 27.8 Inositol 100 Vitamin B1 0.1 Vitamin B6 0.5 Nicotinic acid 0.5 Glycine 2

[0039] Table 2 Composition of B5 medium

[0040]

[0041]

[0042] Table 3 Composition of Miller medium

[0043]

[0044]

[0045] Example 1

[0046] An embodiment of the method for inducing white peony leaf begonia unpollinated ovule callus according to the application comprises the following steps:

[0047] (1) The female flowers of white peony leaf begonia with normal appearance quality, no diseases and pests, and about to open for about 1-3 days are picked up between 8 am and 14 pm, and are placed at 4℃ for pretreatment for 6 days;

[0048] (2) Put the explant in a sterile empty bottle, first wash it with sterile water for 1-2 times, then immerse it in 75% ethanol for 20s on the super-clean workbench, wash it with sterile water for 1-2 times, immerse it in 2% sodium hypochlorite solution for 20min, and finally wash it with sterile water for 5-6 times;

[0049] (3) After sterilization, the white peony leaf begonia pistil is dried with sterilized filter paper, the perianth of the pistil is cut off with a scalpel, the ovary carpel is removed with a sharp forceps, and then the ovary carpel is rolled with a blunt forceps and adhered to the culture medium, and finally sealed with sealing film. The sealed culture dish is placed in a culture room for culture, and the culture conditions are as follows: the temperature is 23-27℃, the light time is 16 hours / day, the air relative humidity is 30%-65%, and the culture time is 90 days.

[0050] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; and the pH value of the induction medium is 5.8-6.0.

[0051] The basic medium is a Miller induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0052] The concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.05:0.25:0.25.

[0053] The weight ratio of the sucrose and agar is sucrose:agar=30:8.

[0054] The preparation method of the induction medium is as follows: the plant growth regulator, sucrose and agar are weighed according to the proportion, and the rest is supplemented with the basic medium; after the induction medium is prepared, high-pressure sterilization is carried out at a pressure of 105Kpa and a temperature of 121℃ for 20min, and after sterilization, the induction medium is divided into culture dishes on the super-clean bench.

[0055] Example 2:

[0056] The white peony leaf begonia unpollinated ovule callus induction method provided by the application is an embodiment of the white peony leaf begonia unpollinated ovule callus induction method.

[0057] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; and the pH value of the induction medium is 5.8-6.0.

[0058] The basic medium is a Miller induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0059] The concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.05:0.50:0.50.

[0060] The weight ratio of the sucrose and agar is sucrose:agar=30:8.

[0061] Embodiment 3

[0062] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the application; the difference between this embodiment and embodiment 1 is that:

[0063] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; the pH value of the induction medium is 5.8-6.0.

[0064] The basic medium is Miller induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0065] The concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.05:1.00:1.00.

[0066] The weight ratio of the sucrose and agar is sucrose:agar=30:8.

[0067] Embodiment 4

[0068] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the application; the difference between this embodiment and embodiment 1 is that:

[0069] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; the pH value of the induction medium is 5.8-6.0.

[0070] The basic medium is Miller induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0071] The concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.07:0.25:0.50.

[0072] The weight ratio of the sucrose and agar is sucrose:agar=30:8.

[0073] Embodiment 5

[0074] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the application; the difference between this embodiment and embodiment 1 is that:

[0075] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; the pH value of the induction medium is 5.8-6.0.

[0076] The basic medium is a Miller induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0077] The concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.07:0.50:1.00.

[0078] The weight ratio of the sucrose and agar is sucrose:agar=30:8.

[0079] Embodiment 6

[0080] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the application; the difference between this embodiment and embodiment 1 is that:

[0081] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; the pH value of the induction medium is 5.8-6.0.

[0082] The basic medium is a Miller induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0083] The concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.07:1.00:0.25.

[0084] The weight ratio of the sucrose and agar is sucrose:agar=30:8.

[0085] Embodiment 7

[0086] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the application; the difference between this embodiment and embodiment 1 is that:

[0087] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; the pH value of the induction medium is 5.8-6.0.

[0088] The basic medium is a Miller induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0089] The concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.10:0.25:1.00.

[0090] The weight ratio of the sucrose and the agar is sucrose: agar = 30:8.

[0091] Embodiment 8

[0092] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the present application; the difference between this embodiment and embodiment 1 is that:

[0093] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; the pH value of the induction medium is 5.8-6.0.

[0094] The basic medium is Miller induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0095] The concentration ratio of the TDZ, the NAA and the 6-BA is TDZ:NAA:6-BA = 0.10:0.50:0.25.

[0096] The weight ratio of the sucrose and the agar is sucrose: agar = 30:8.

[0097] Embodiment 9

[0098] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the present application; the difference between this embodiment and embodiment 1 is that:

[0099] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; the pH value of the induction medium is 5.8-6.0.

[0100] The basic medium is Miller induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0101] The concentration ratio of the TDZ, the NAA and the 6-BA is TDZ:NAA:6-BA = 0.10:1.00:0.50.

[0102] The weight ratio of the sucrose and the agar is sucrose: agar = 30:8.

[0103] Embodiment 10

[0104] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the present application; comprising the following steps:

[0105] (1) picking up the Begonia fimbristipula female flowers with normal appearance quality, no disease and insect pests, and about to open for about 1-3 days, and placing them in 4℃ for 2 days of pretreatment;

[0106] (2) Place the explant in a sterile empty bottle, rinse with sterile water 1-2 times, then soak in 75% ethanol for 20 seconds on a clean bench, rinse with sterile water 1-2 times, soak in 2% sodium hypochlorite solution for 20 minutes, and finally rinse with sterile water 5-6 times.

[0107] (3) After sterilization, dry the female flowers of Angelica dahurica and Begonia florida with sterile filter paper. Cut off the perianth segments of the female flowers with a scalpel, then remove the carpels of the ovary with sharp forceps. Use blunt forceps to hold the ovules in the induction medium and roll them to make them adhere to the medium. Finally, seal the ovules with sealing film. Place the sealed petri dishes in a culture room for cultivation. The cultivation conditions are: temperature 23-27℃, light time 16 hours / day, relative humidity 30%-65%, and cultivation time 60-90 days.

[0108] The induction medium includes: basal medium, plant growth regulator, carbon source and coagulant; the pH value of the induction medium is 5.8-6.0.

[0109] The basal culture medium is Miller induction medium; the plant growth regulators include TDZ, NAA and 6-BA; the carbon source is sucrose; and the solidifying agent is agar.

[0110] The concentration ratio of TDZ, NAA and 6-BA is TDZ:NAA:6-BA = 0.07:0.50:0.50.

[0111] The weight ratio of sucrose to agar is 30:8.

[0112] The induction medium is prepared as follows: plant growth regulator, sucrose and agar are weighed according to the proportion, and the remainder is supplemented with basic culture medium; after the induction medium is prepared, it is autoclaved at a pressure of 105 kPa and a temperature of 121°C for 20 min, and after sterilization, it is dispensed into petri dishes on a clean bench.

[0113] Example 11:

[0114] An embodiment of the method for inducing callus tissue from unpollinated ovules of Begonia dahurica leaves according to the present invention includes the following steps:

[0115] (1) Pick female flowers of Angelica dahurica that are in normal appearance and free from pests and diseases, and are about to open in about 1-3 days, between 8 am and 2 pm, and place them at 4℃ for 2 days for pretreatment.

[0116] (2) the explant is placed in a sterile empty bottle, first washed with sterile water for 1-2 times, then soaked with 75% ethanol for 20s on the super-clean workbench, washed with sterile water for 1-2 times, soaked with 2% sodium hypochlorite solution for 20min, and finally washed with sterile water for 5-6 times;

[0117] (3) the sterilized white peony leaf begonia pistil is dried with sterilized filter paper, the perianth of the pistil is cut off with a surgical knife, the ovary carpel is removed with a sharp forceps, and the ovary carpel is rolled with a blunt forceps to make the ovule adhere to the culture medium, and finally sealed with sealing film. The sealed culture dish is placed in a culture room for culture, and the culture conditions are as follows: the temperature is 23-27℃, the light time is 16 hours / day, the air relative humidity is 30%-65%, and the culture time is 60-90 days.

[0118] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant; and the pH value of the induction medium is 5.8-6.0.

[0119] The basic medium is a B5 induction medium; the plant growth regulator comprises TDZ, NAA and 6-BA; the carbon source is sucrose; and the coagulant is agar.

[0120] The concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.07:0.50:0.50.

[0121] The weight ratio of the sucrose and agar is sucrose:agar=30:8.

[0122] The preparation method of the induction medium is as follows: the plant growth regulator, sucrose and agar are weighed according to the proportion, and the rest is supplemented with the basic medium; after the induction medium is prepared, high-pressure sterilization is carried out at a pressure of 105Kpa and a temperature of 121℃ for 20min, and after sterilization, the induction medium is divided into culture dishes on the super-clean bench.

[0123] Example 12:

[0124] One embodiment of the method for inducing unpollinated ovule callus of white peony leaf begonia according to the present application;

[0125] The difference between the present embodiment and example 10 is that the 4℃ pretreatment is not carried out.

[0126] Example 13:

[0127] One embodiment of the method for inducing unpollinated ovule callus of white peony leaf begonia according to the present application;

[0128] The difference between the present embodiment and example 10 is that the 4℃ pretreatment is not carried out.

[0129] Example 14

[0130] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the present application;

[0131] The difference between this example and example 10 is that the 4℃ pretreatment lasts for 6 days.

[0132] Example 15

[0133] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the present application;

[0134] The difference between this example and example 10 is that the 4℃ pretreatment lasts for 6 days.

[0135] Example 16

[0136] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the present application;

[0137] The difference between this example and example 14 is that the high-temperature heat shock lasts for 4 days.

[0138] Example 17

[0139] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the present application;

[0140] The difference between this example and example 14 is that the high-temperature heat shock lasts for 6 days.

[0141] Comparative Example 1

[0142] An embodiment of the method for inducing unpollinated ovule callus of Begonia fimbristipula according to the present application; comprising the following steps:

[0143] (1) Pick the Begonia fimbristipula female flowers with normal appearance quality, no disease and insect pests, and about to open in about 1-3 days at 8 am to 14 pm, and place them in 4℃ for 2 days of pretreatment;

[0144] (2) Place the explants in a sterile empty bottle, first rinse 1-2 times with sterile water, then soak in 75% ethanol for 20s on the clean bench, rinse 1-2 times with sterile water, soak in 2% sodium hypochlorite solution for 20min, and finally rinse 5-6 times with sterile water;

[0145] (3) After the sterilized female flower of P. multifida is dried with sterilized filter paper, the perianth of the female flower is cut off with a surgical knife, and then the ovary carpel is removed with a sharp forceps, and the ovary carpel is rolled with a blunt forceps to make the ovule stick to the induction medium, and finally the induction medium is sealed with sealing film. The sealed culture dish is placed in a culture room for culture, and the culture condition is that the temperature is 23-27℃, the light time is 16 hours / day, the air relative humidity is 30%-65%, and the culture time is 60-90 days.

[0146] The induction medium comprises a basic medium, a plant growth regulator, a carbon source and a coagulant, and the pH value of the induction medium is 5.8-6.0.

[0147] The basic medium is MS induction medium, the plant growth regulator comprises TDZ, NAA and 6-BA, the carbon source is sucrose, and the coagulant is agar.

[0148] The concentration ratio of the TDZ, NAA and 6-BA is TDZ:NAA:6-BA=0.07:0.50:0.50.

[0149] The weight ratio of the sucrose and agar is sucrose:agar=30:8.

[0150] The preparation method of the induction medium is that the plant growth regulator, sucrose and agar are weighed according to the proportion, and the rest is supplemented with the basic medium; after the induction medium is prepared, high-pressure sterilization is carried out under the condition that the pressure is 105Kpa and the temperature is 121℃ for 20min, and after sterilization, the induction medium is divided into culture dishes on the super-clean bench.

[0151] Experimental results

[0152] 1. Effect of different plant growth regulator proportions on induction of callus

[0153] The female flower of P. multifida is treated according to the induction method of embodiments 1-9, and the induction rate is calculated after 90 days of culture, and the results are shown in Table 4, and the calculation formula of the induction rate is as follows (the number of contaminated dishes is excluded):

[0154] Induction rate=(number of dishes producing callus / number of dishes of inoculated ovules)×100%

[0155] Table 4. Effect of different plant growth regulator proportions on induction rate

[0156]

[0157]

[0158] From Table 4, it can be seen that different combinations of plant growth regulators affect the induction rate of the unpollinated ovule callus of B. japonica. In the above examples, the induction medium is the Miller induction medium based medium, and the induction medium is added with 0.10 mg / L TDZ, 1.0 mg / L NAA and 0.5 mg / L 6-BA. The induction rate of the unpollinated ovule callus of B. japonica is the highest, and the callus induction rate is 58.33%. In addition, by comparing the callus induction rates of the three plant growth regulators, it is found that the order of the hormones affecting the induction of the unpollinated ovule callus of B. japonica is NAA > TDZ > 6-BA.

[0159] 2. Effect of different basic media on callus induction

[0160] According to Examples 10-11, the induction method of Comparative Example 1 is used to treat the female flowers of B. japonica, and the induction rate is calculated after 60-90 days of culture. The results are shown in Table 5, and the calculation formula of the induction rate is as follows (excluding the number of contaminated dishes):

[0161] Induction rate = (number of dishes producing callus / number of dishes of ovules inoculated) x 100%

[0162] Table 5 Effect of different basic media on induction of unpollinated ovule callus of B. japonica

[0163]

[0164]

[0165] The MS medium is a high-salt component medium, which is characterized by high inorganic salt concentration, and contains high nitrogen, potassium, ammonium salt and nitrate; the B5 medium is a high potassium nitrate content medium, which contains low ammonium salt, high nitrate and thiamine hydrochloride (vitamin B1). The Miller medium is a medium with medium inorganic salt content, and compared with the MS medium, the inorganic element content is reduced by 1 / 3-1 / 2, the type of trace elements is reduced, and the inositol is not contained. The test results in Table 5 show that the callus induction rate of the Miller medium used in Example 10 is significantly higher than that of the MS medium of Comparative Example 1 and the B5 medium of Example 11 on the unpollinated ovule of B. japonica, and it can be inferred that the medium with lower inorganic salt concentration is more suitable for the ovule culture of B. japonica. Therefore, among the three induction media, the Miller medium is the best basic medium for ovule culture.

[0166] 3. Effect of different induction treatment methods on induction and redifferentiation

[0167] The female flowers of Begonia fimbristipula were treated by the induction method of Examples 12-17, and the induction rate was calculated after 60 days of culture. The results are shown in Table 6, and the calculation formula of the induction rate is as follows (excluding the number of contaminated dishes):

[0168] Induction rate = (the number of dishes producing callus / the number of dishes of inoculated ovules) x 100%

[0169] After the callus was induced, the callus was subjected to differentiation culture.

[0170] The differentiation induction medium was prepared:

[0171] Combination A: MS medium, 30 g / L sucrose, 7 g / L agar, 0.5 mg / L 6-BA, 0.2 mg / L NAA;

[0172] Combination B: MS medium, 30 g / L sucrose, 7 g / L agar, 0.5 mg / L 6-BA, 0.5 mg / L NAA;

[0173] Combination C: MS medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-BA, 0.2 mg / L NAA;

[0174] Combination D: MS medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-BA, 0.5 mg / L NAA; pH 5.8-6.0, and after dispensing into culture bottles, high-pressure sterilization was performed at a pressure of 105 Kpa and a temperature of 121°C for 20 min.

[0175] The undifferentiated and well-grown callus after low-temperature treatment and heat shock treatment was inoculated in the differentiation induction medium, 15 calli per bottle. The culture conditions were: temperature 23-27°C, light time 16 hours / day, air relative humidity 30%-65%, and culture time 60 days. The differentiation rate was calculated, and the calculation formula is as follows (excluding the number of contaminated calli), and the results are shown in Table 6.

[0176] Differentiation rate = (the number of differentiated calli / the number of inoculated calli) x 100%

[0177] Table 6 Effect of different pretreatment methods on callus induction and redifferentiation of unpollinated ovules of Begonia fimbristipula

[0178]

[0179] From the above table, different pretreatment methods have no great difference in induction rate, but in the differentiation culture of callus, heat shock treatment for 4-6 days can further improve the differentiation rate compared with other treatments. Among them, the callus differentiation rate of heat shock treatment for 6 days of white arisaema leaf begonia unpollinated ovule is the highest, which is 2.22%.

[0180] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. An induction culture medium, characterized in that, The induction medium includes a basal medium, a plant growth regulator, a carbon source, and a coagulant; the basal medium includes either B5 medium or Miller medium; the plant growth regulator is composed of TDZ, NAA, and 6-BA; the concentration ratio of TDZ, NAA, and 6-BA is TDZ:NAA:6-BA = (0.05-0.10):(0.25-1.00):(0.25-1.00); the pH of the induction medium is 5.8-6.0; the carbon source is sucrose; the coagulant is agar; the weight ratio of sucrose to agar is sucrose:agar = 30:

8.

2. The induction culture medium as described in claim 1, characterized in that, The basal culture medium is Miller medium.

3. The induction culture medium as described in claim 1, characterized in that, The concentration ratio of TDZ, NAA, and 6-BA is TDZ:NAA:6-BA = 0.10:(0.5-1.00):(0.25-0.50).

4. The application of the induction culture medium as described in any one of claims 1-3 in inducing callus tissue from unpollinated ovules of Begonia dahurica.

5. A method for inducing callus from unpollinated ovules of Begonia dahurica, characterized in that, After cleaning and disinfecting the explants, they are inoculated onto the induction culture medium described in any one of claims 1-3, the culture medium is sealed, and cultured to obtain the callus tissue; the explants are fresh female flowers of Begonia dahurica leaves 1-3 days before opening.

6. The method for inducing callus from unpollinated ovules of Begonia dahurica leaf as described in claim 5, characterized in that, The explants are pretreated before culture; the pretreatment method is to place the explants at 4°C for 2-6 days or place the sealed culture medium at 35°C for 2-6 days before washing.

7. The method for inducing callus from unpollinated ovules of Begonia dahurica leaf as described in claim 5, characterized in that, The explants were cleaned with sterile water; the disinfection process involved first soaking them in ethanol, then rinsing them with sterile water, then soaking them in a 2% sodium hypochlorite solution, and finally rinsing them with sterile water.

8. The method for inducing callus from unpollinated ovules of Begonia dahurica leaf as described in claim 5, characterized in that, The cultivation conditions are: temperature 23-27℃, light duration 16h / day, relative humidity 30%-65%, and cultivation time 60-90 days.

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